Abstract: Androgen dysregulation contributes to disorders such as irritable bowel syndrome (IBS), polycystic ovarian syndrome (PCOS), and prostate cancer. Inactivation of androgens occurs via glucuronidation by host UDP-glucuronosyltransferases, enhancing their polarity and excretion into the intestines. However, microbial β-glucuronidase (GUS) enzymes in the gut can reactivate these androgens by removing the glucuronide, allowing them to engage local receptors, re-enter circulation, and undergo enterohepatic recirculation. We hypothesize that reduced GUS abundance correlates with androgen-deficient conditions. GUS enzymes are structurally diverse, with subfamilies exhibiting distinct substrate preferences. Our studies confirm that Loop 1 GUS proteins, such as E. coli GUS, efficiently process testosterone-glucuronides. Ongoing research integrates structural biology and bioanalytical approaches to further characterize these enzymes and assess their distribution in metagenomic data from mouse models and human subjects. Collectively, our work aims to elucidate the role of gut microbial GUS enzymes in androgen reactivation and their potential impact on intestinal diseases linked to steroid hormone imbalances.